Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long-term use of this excellent pain reliever in man is limited by side-effects that include analgesic tolerance and opioid-induced bowel dysfunction, with constipation being the most common and debilitating symptom. The long-term goals of this study are to elucidate the mechanisms that lead to tolerance to many of the effects of opioids, including slowing of gastrointestinal transit but not constipation. The main hypothesis to be tested is that differences in the cellular signaling properties of morphine determine the development of tolerance in the ileum but not the colon. Preliminary data suggest that morphine tolerance in the ileum is associated with an uncoupling of the ? opioid receptor from its downstream signaling proteins. Unlike the ileum, the colon which is the major site for constipation does not develop tolerance to repeated administration of morphine. The major objective of specific aim 1 is to test the hypothesis that down-regulation of ? arrestin2 is associated with morphine tolerance in the ileum. The specific goals are to characterize the concentration and temporal relationship for ? arrestin 2 downregulation and tolerance development. Functional and biochemical studies will be utilized to correlate the effect of chronic morphine in-vitro and in-vivo utilizing ?-arrestin2 knock-out mice. Specific Aim 2 will test the hypothesis that ? arrestin2 acts as a scaffolding protein to regulated downstream signaling including MAP kinase, Src kinase, Akt and protein kinase C. Preliminary findings suggest that unlike morphine induced antinociceptive tolerance, in the ileum downregulation of phospho-ERK correlates with tolerance development suggesting fundamental differences in the mechanism for opioid tolerance in the gastrointestinal tract from CNS. This aim will also examine if downregulation of ? arrestin 2 is mediated via altered ubiquitination. Specific Aim 3 will explore the effect of long-term morphine on isolated enteric neurons from the adult mouse myenteric plexus. In this aim, single enteric neurons from the colon and ileum will be characterized and tested to determine morphine- induced changes in electrical excitability and effects on sodium, calcium and potassium channels in wild-type and ? arrestin2 knock-out mice. The information obtained from these studies will increase our understanding of the mechanisms of opioid tolerance and ultimately physical dependence in the gastrointestinal tract and in the brain.